Frankia bacteria, actinorhizal plant symbiosis, plays an important role in the colonization of soils contaminated with toxic aromatic hydrocarbons. Understanding of bacteria in actinorhizal symbiosis is greatly facilitated by the availability of sequenced genomes. Analysis of these frankia genomes revealed that these bacteria are metabolically diverse and have the potential for toxic aromatic hydrocarbon degradation.
Phenol Destruction of Frankia Bacteria
Phenol or hydroxybenzene consists of a benzene ring substituted with a hydroxyl group. Derivatives of this molecule are popularly known as phenolic compounds. Phenolic compounds are ubiquitous chemicals with a variety of properties and uses. Phenol, the simplest phenolic compound, is widely used in petroleum and coal processing, tinctorial and metallurgical industries, and many other industrial applications. It also enters the environment through vehicle exhaust and as a product of natural metabolic processes, chlorophenols are widely used as biocides in agricultural applications.
While anthropogenic phenolics are generally dangerous, compounds that are natural are mostly harmless in concentrations found in foods such as coffee and tea, and some are used as antibiotics. However, the toxicity of some phenolics, especially phenol and chlorinated phenols, has led to significant research activities devoted to phenol enhancement. Acute and chronic exposure to phenol and chlorophenol has serious health effects. It causes lipid peroxidation, which ultimately leads to tissue necrosis, liver and kidney damage. Additionally, chlorophenol exposure is associated with an increased risk of cancer, immune deficiencies, and teratogenic effects.
General Phenol Degradation Pathway
One of the most promising techniques for removing anthropogenic phenolics from the environment is bioremediation. Most bacteria degrade phenolics using catechol catabolic enzymes, most notably catechol-2,3-dioxygenase. The phenols are first hydroxylated to form catechol, and then the catechol-2,3-dioxygenase cleaves the benzene ring at the meta position. Therefore, the degradation pathway that starts with catechol-2,3-dioxygenase is called the meta pathway. Although the metabolism is the most common, its degradation can begin with cleavage in the circulation or ortho position using catechol-1,2-oxygenase.
After ring cleavage, the 2-hydroxymeuconic semialdehyde hydrolase catalyzes a decarboxylation reaction yielding 4-oxalocrotonate. 4-oxalocrotonate is hydrated with 2-oxopent-4-enoate hydratase to form 4-hydroxy-2-oxovalerate. The 4-hydroxy-2-oxovalerate aldolase then cleaves 4-hydroxy-2-oxovalerate into pyruvate and acetaldehyde, which can then be incorporated into central metabolic pathways.
Frankia Bacteria and Phenolic Compounds
Frankia spp both produce and are affected by phenolic compounds, but they are uncertain and can degrade frankia phenol and other phenolic compounds. Frankia phenolics were first studied in the context of plant-microbe interactions. Despite the distinct functional and morphological similarities between nodules and legumes, the molecular and physiological mechanisms that control nodulation are different. As such, it is still an area of intensive research in terms of its unique nodulation process. Alnus spp (Alder) plants have unusually high levels of phenolics in frankia and also root exudates that affect their growth.
The alnus phenolics tested inhibit frankia growth to varying degrees. Specifically, benzoic acids are less inhibitory than cinnamic acids such as caffeine. However, a plant branches into phenolic, o-hydroxyphenylacetic acid, frankia overgrowth, and both benzoic and cinnamic acids. Low concentration plant phenolics, as well as frankia gene expression, high concentrations simply inhibit biosynthesis. Interestingly, it also increases the phenolic expression of the host plant, causing it to produce more phenols, flavonoids, and hydroxycinnamic acids.
It can promote the excretion of phenolics as a way to increase available nutrients. However, frankia depends on its ability to reduce phenolic compounds. Although no studies have proven this, there is genetic evidence that when it degrades phenolic compounds, this bacterium may have the ability to degrade phenolic substances. First, some strains have genes encoding the production of catechol and other phenolic compounds. Since bacteria usually rescue the biomolecules they produce, the presence of an anabolic pathway suggests that a catabolic pathway is also present. Additionally, multiple strains contain catechol-2,3-dioxygenase, the most important enzyme in the phenol degradation pathway. Rhodococcus spp, a closely related bacterium, uses the catechol-2,3-dioxygenase pathway to grow with phenol as its sole carbon source. The same strain is also able to break down the more stubborn pentachlorophenol through circulation. This suggests that Frankia can break down phenol, a property that can be applied in bioremediation efforts. Several Frankia strains can thrive on phenol, quercetin, catechol, and other phenolic compounds, but the metabolism of their breakdown has not been studied.
Frankia and Naphthalene Degradation
Naphthalene is a ubiquitous polyaromatic hydrocarbon composed of two benzene rings joined at 9 and 10 carbons. Naphthalene is produced by the distillation and crystallization of coal tar, as well as a byproduct of fossil fuel combustion and cigarette smoke. It is used in a number of industrial applications, including as feedstock for the production of plastics and resins and as a component of creosote-based wood preservatives. Naphthalene is also used in the tincture and leather tanning industries.
Unlike many organic pollutants, it does not bioaccumulate but instead is metabolized and excreted through the urine. However, as naphthalene is a problematic contaminant with multiple toxic effects, acute exposure causes hemolytic anemia, liver and neurological damage. It is also associated with an increased risk of cancer when exposed to excessive amounts. The toxicity of naphthalene and its prevalence as a pollutant spurred research on remediation techniques, including bioremediation and biodegradation.
The mothballs biodegradation pathway was first studied in one strain, and also has two degradation pathways, upper and lower, of naphthalene associated with pseudomonas bacteria. The upper pathway catabolizes naphthalene to produce salicylate and a pyruvate molecule, while the lower path separates salicylate into acetyl Co-A and pyruvate. The first step of the upper pathway is catalyzed by four proteins and they are as follows:
Naphthalene dioxygenase reductase,
• Naphthalene dioxienase ferredoxin,
• Naphthalene dioxienase
Fe-S protein small and large subunits,
This enzyme collection is dehydrogenated with cis-naphthalene dihydrodiol, then naphthalene oxidizes cis-dihydodiol dehydrogenase to form 1,2-dihydroxy naphthalene.
Naphthalene Degradation in France
Frankia metabolizes naphthalene in a related way as a single source of carbon and energy. In particular, it uses the protochactate pathway to convert naphthalene or a derivative into acetyl Co-A and succinyl Co-A. This finding was found in the naphthalene degradation suggested in previous field studies. Frankia, in its symbiosis with alder, increases polyaromatic hydrocarbon degradation in the first 1.5 years in oil-sand waste, and shows equal naphthalene degradation after 2.5 years. Alder symbiosis develops in PAH-contaminated areas. Interestingly, alder plants found in areas contaminated with these PAHs, strain 3 as opposed to frankia normal strain 1, suggesting that this contaminant affects tubing or survival of actinorhizal plants. Taken together, these findings suggest that frankia mothballs are a useful tool in healing.
Under oxygen conditions, microbial degradation of the ß-ketoadipate pathway of many aromatic compounds via the catechol or protocatechate branch, cleavage by ortho catechol 1,2-dioxygenase and protocat 3,4-dioxygenase, or meta-catechol-2,3-dioxygenase and protocatechate-4. It takes place by cleavage with 5-dioxygenase.
Potential Protocatechate Degradation Pathway in Frankiada
Besides Frankia QA3, which is the protochatechuate pathway, several other potential protocol pathways have been identified from bioinformatics analysis of existing Frankia genomes. In Frankia EuI1c, a potential operon for a putative protocol fire path is defined. This operon encodes predictive gene products involved in the putative pathway, including protocatechate 3,4-dioxygenase alpha and beta subunits, fumarate lyase, 3-oxoadipate enol-lactonase, and 4-hydroxybenzoate 3-monooxygenase. These gene products are similar to the protocathecate degradation pathway found in it. According to the results obtained, after the frankia has been converted into protocathecate, it can use the protocatechate degradation pathway to degrade many aromatic ring compounds.
Petroleum-based energy and products are widely used around the world. The prevalence of oil inevitably leads to serious environmental pollution. Petroleum is a complex mixture of more complex chemicals such as hydrocarbons, cycloalkanes, aromatic hydrocarbons and asphaltenes. These chemicals and their derivatives, called petrogenic compounds, are released into the environment as a result of oil spills and combustion of petroleum-based products. Oil spills are one of the most serious sources of oil pollution, as well as destroying water and marine environments. Ongoing research is important to identify new methods for oil recovery. Because oil spills and other types of pollution from oil continue to pose environmental health risks.
Bacteria and fungi that break down hydrocarbons are common in marine and freshwater environments, as well as in soil habitats. The pseudomonacalkane hydroxylase (monooxygenase) system consists of three components: alkane hydroxylase (AlkB), rubredoxin and rubredoxin reductase. This system is responsible for the first oxidation step in the use of n-alkanes. Similar alkane hydroxylase systems have been found in various alkane reducing bacteria. The 2B5 strain reduces C13 – C30 n-alkanes and branched alkanes (pure and phytan) from crude oil as the sole carbon source via a new alkane hydroxylase gene. Other acinetobacteria can use n-alkanes with chain lengths of C10 – C40 as their sole carbon source. Additionally, Rhodococcus strains are characterized in the presence of more than two alkane hydroxylase in two, and both organisms are at least four alkane monooxygenase gene homologs.
A bioinformatics approach has been used to identify these potential hydrocarbon degradation pathways among those sequenced frankia strains. Identify genes, frankia genome database and potential pathways analyzed functionally for known hydrocarbon degradation pathways. According to the preliminary data of the study, the F. alni ACN14a genome is one of the known enzymes involved in the degradation of n-alkanes into a putative alkan-1 monooxygenase gene. Moreover, a similar gene frankia sp was also found in the EAN1pec genome, and these bioinformatics results support the hypothesis. Frankia can degrade hydrocarbons from oil spills, however, these preliminary results need to be investigated further.
Studies linking metabolic capacity with gene function are the first step in using bacteria for their bioremediation abilities. More bioinformatics data mining is required to uncover the unmatched metabolic potential. However, these studies in silico require laboratory experiments to validate these capabilities. According to limited field studies, actinorhizal nodule invasion appears to be controlled by environmental conditions. The presence of Frankia lineage 3 strains found within alder nodules in PAH-stressed soils suggest that this lineage may have a greater metabolic potential. The larger genome size of this strain compared to other infective strains also supports this hypothesis. However, further studies are needed to confirm this assumption.
Writer: Ozlem Guvenc Aggoglu